Persistent bacterial infections are a major cause of death in cystic fibrosis patients and immune-compromised individuals. A number of gram-negative bacteria including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis, cause infections that are difficult to treat because the bacteria form a """"""""biofilm community"""""""" that renders them less sensitive to traditional antibiotics. Quorum sensing, mediated by acylhomoserine lactone (AHL) signaling molecules, regulates pathogenesis and biofilm formation in P. aeruginosa. Therefore, understanding the molecular basis of quorum sensing is a high priority in the development of novel anti-bacterial agents. The long term goal of this project is to extend the understanding of the quorum-sensing system to the atomic level to develop a detailed description of the mechanisms that control bacterial pathogenesis. The main focus of this proposal is the class of enzymes that produce the AHL signal, AHL-synthases, because bacteria lacking the AHL signal fail to become pathogenic or form stable biofilms. Although there are models of the mechanism of action of the AHL-synthases, there are currently no structures of any AHL synthase. High resolution structural information is absolutely essential for fully understanding the mechanism of AHL synthesis and will provide the basis for future structure-based inhibitor design for development of novel therapeutics.
The specific aims for this project are: (I) determine the high resolution crystal structure of the Pantoea stewartii subsp. Stewartii AHL-synthase (EsaI) to understand its function, mechanism, and relationship to other enzymes that utilize similar substrates. Perform mutagenesis, binding and kinetics experiments with EsaI to better understand the catalytic mechanism and substrate specificity. (II) Study the P. aeruginosa AHL-synthase, LasI, using structural and biochemical techniques to understand how specificity of AHL production is determined. (III) Establish whether the AHL-synthase homologues in divergent organisms produce a homoserine lactone signal using mass spectrometry and activity assays. Study the structures and mechanisms to determine similarities to other AHL synthases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI048660-01A1
Application #
6399559
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Korpela, Jukka K
Project Start
2001-07-01
Project End
2006-05-31
Budget Start
2001-07-01
Budget End
2002-05-31
Support Year
1
Fiscal Year
2001
Total Cost
$334,058
Indirect Cost
Name
University of Colorado Denver
Department
Pharmacology
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
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Tizzano, Marco; Gulbransen, Brian D; Vandenbeuch, Aurelie et al. (2010) Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals. Proc Natl Acad Sci U S A 107:3210-5
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Duerkop, Breck A; Herman, Jake P; Ulrich, Ricky L et al. (2008) The Burkholderia mallei BmaR3-BmaI3 quorum-sensing system produces and responds to N-3-hydroxy-octanoyl homoserine lactone. J Bacteriol 190:5137-41
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Khan, Sharik R; Herman, Jake; Krank, Jessica et al. (2007) N-(3-hydroxyhexanoyl)-l-homoserine lactone is the biologically relevant quormone that regulates the phz operon of Pseudomonas chlororaphis strain 30-84. Appl Environ Microbiol 73:7443-55
Kirwan, J Paul; Gould, Ty A; Schweizer, Herbert P et al. (2006) Quorum-sensing signal synthesis by the Yersinia pestis acyl-homoserine lactone synthase YspI. J Bacteriol 188:784-8
Gould, Ty A; Herman, Jake; Krank, Jessica et al. (2006) Specificity of acyl-homoserine lactone synthases examined by mass spectrometry. J Bacteriol 188:773-83

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